Glycerol permeability in erythrocytes from an amphibian that accumulates glycerol in the cold

Abstract

During cold acclimation, gray treefrogs Hyla chrysoscelis accumulate >50 mM glycerol in the extracellular fluid. That glycerol likely is synthesized in the liver and then delivered to other tissues, where it acts as an intracellular cryoprotectant and osmotic agent. We hypothesized that the permeability of cells to glycerol would be enhanced in cold acclimation. We tested this hypothesis on erythrocytes using two protocols, a lysis assay in which glycerol uptake induces osmotic lysis, and uptake of radio-labeled glycerol. We addressed four questions: a) What is the effect of temperature on glycerol permeability? (Permeability was markedly reduced in cells tested at lower temperatures.) b) What is the effect of cold acclimation on permeability properties? (We were unable to detect a change in permeability induced by cold-acclimation.) c) Is there geographic variation in these parameters? (Frogs from southern populations had similar properties as those from Ohio.) d) Are aquaporins involved in the uptake of glycerol? (Both lysis induced by glycerol uptake and influx of labeled glycerol were essentially abolished by mercurial compounds that inhibit aquaporins. We detected one prominently expressed aquaporin in erythrocytes, identified using primers designed from an ortholog likely to be in the glyceroporin family.) Thus, we suggest that treefrog red blood cells have glyceroporins that are constitutively expressed, and that allow intracellular accumulation of glycerol during cold acclimation.